Fabrication and characterization of DNA–thionine–carbon nanotube nanocomposites

https://doi.org/10.1016/j.jelechem.2008.03.020Get rights and content

Abstract

The DNA–thionine–carbon nanotube (DNA–Th–CNT) nanocomposites were prepared by immobilizing DNA on the surface of carbon nanotubes (CNTs) via thionine (Th). The fabrication process was characterized by Raman spectroscopy, UV–vis spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM). The results indicated that Th could facilitate the immobilization of DNA molecules onto the surface of CNTs, and DNA retained its native secondary conformational structure after it was immobilized. These demonstrated that Th may be served as an effective mediator for immobilization of DNA on CNTs. The nanocomposites were immobilized on the surface of glassy carbon (GC) electrode forming the DNA–Th–CNTs/GC electrode. The electrode was used to study the interaction between Co(bpy)33+ probe and the immobilized DNA by cyclic voltammetry, whose results indicated that Co(bpy)33+ could be electrostatically bound onto the immobilized DNA. The method of the present work has the advantages of rapid and facile CNTs functionalization, high immobilization efficiency. The nanocomposites have a good electrochemical response with a long-term stability, suggesting they have the potential use in the fields of DNA biosensors.

Introduction

Electrochemical sensors for determining DNA sequences are of central importance to diagnosis and treatment of genetic diseases [1], [2], [3], [4]. The high sensitivity of such devices, as well as portability, low cost, minimal power requirements make them suitable candidates for obtaining sequence-specific information in clinical [5], environmental [6] and forensic investigations [7].

Recent works on DNA sensor mainly focus on DNA hybridization and its damage, several review articles have been published in this field [8], [9], [10], [11], [12]. Three steps were usually included in electrochemical detection of DNA hybridization: (a) immobilization of a DNA probe onto a transducer surface; (b) hybridization of the probe with target DNA; and (c) subsequent electrochemical detection. Among the three steps, the immobilization and electrochemical detection are two key steps [13], [14], [15]. The electrochemical detection usually realized via the direct oxidation of the guanine and/or adenine. However, the oxidation of purine at a solid electrode actually happens at higher potential (usually higher than 1 V), at which some easily oxidation species can interference the detection [16], [17], [18], [19], [20]. To overcome this problem, the redox indicators, such as methylene blue [9], [11], [21], [22], metal complexes [9], [11], [22], [23], [24], [25], and ferrocene and its derivates [9], [11] etc. were used. These indicators usually exhibited a high reversibility and low redox potentials (around 0 V). For DNA immobilization on transducer surface, many strategies such as adsorption [26], [27], [28], [29], covalent bonding [30], [31], [32], [33], physical entrapping co-polymerization [34], [35], [36], and via the advin–biotin affinity system [37], [38] etc. have been developed. Among those, adsorption is very attractive method due to its inherent simplicity and accessibility [39], [40]. However, this method usually suffers from low efficiency and stability. Thus, there is still high demand for exploring the new route for DNA immobilization with improved efficiency and stability. Here, we will report a new method for DNA immobilization on the surface of carbon nanotubes (CNTs) via a mediator, thionine (Th).

CNTs are thought to be the result of folding graphite sheets into a seamless shell, exhibit a special sidewall curvature, and possess a π–conjugative structure with a highly hydrophobic surface [41], [42]. These unique properties of the CNTs essentially allow them to interact with many organic compounds, especially polynuclear aromatic compounds, through π–π electronic and/or hydrophobic interactions, and thus forming nanocomposites [43], [44], [45], [46]. Because of the well-defined nanostructure, CNTs can also make a good connection with biomacromolecules (e.g. DNA), therefore, high performance of biosensor can be realized. Due to their subtle electronic characteristic and biocompatibility, CNTs-modified electrodes have been applied to improve electron-transfer reactions of nucleic acid, and to construct electrochemical biosensors for detecting DNA hybridization [47], [48], [49], [50]. Strategies for covalently binding DNA to CNTs by direct carbodiimide coupling or other multistep procedures have been reported. However, these covalently methods often include complicated procedures, resulting low coupling efficiency and destroying the sp2 structures of CNTs [30], [31], [32], [33], [51]. Th is a positively charged redox dye, exhibiting a two-electron and two-proton redox reaction with a high reversibility [52], [53], [54], [55], [56], [57], [58]. The positively charged Th is easily adsorbed on the negatively charged surface of CNTs via the electrostatic interaction and π–π stacking [59], [60]. The adsorption of Th molecules onto the CNTs may also partly neutralize the negative charges of CNTs, which will benefit immobilization of the negatively charged DNA onto CNTs.

In this work, the DNA–Th–CNTs nanocomposites were prepared by immobilizing DNA on the surface of CNTs via Th. The fabrication process was characterized by UV–vis spectroscopy, Raman spectroscopy, AFM, SEM etc. The results indicated that DNA can be immobilized on the surface of CNTs readily with the aid of Th, and exhibiting a high stability. The nanocomposites were then dropped on the surface of glassy carbon (GC) electrode forming the DNA–Th–CNTs/GC electrode. This modified electrode was applied for the study of the interaction between DNA and the known redox indicator, Co(bpy)33+. And this new nanocomposites modified electrode exhibited an improvement of its sensitivity and repeatability compared with the DNA–CNTs electrode.

Section snippets

Reagents

Calf thymus DNA (CT–DNA, Beijing Chemical Reagent, Beijing, China), and Th (Shanghai Chemical Company, Shanghai, China) were used as received. CNTs were purchased from Shenzhen Nanotech Port Ltd. Co. (5–50 nm in diameter, Shenzhen, China). Prior to use, they were treated by refluxing in 3 M HNO3 for 3 h, then were thoroughly washed with water to a neutral state and collected by centrifugation at 18,000 rpm for 15 min. Finally, they were dried under vacuum at 60 °C overnight to obtain purified CNTs.

Characterization

When CNTs suspension was mixed and sonicated with Th solution, the positively charged Th molecule is liable to absorb onto CNTs via positive–negative charge interaction and π–π stacking force forming positively charged Th–CNTs adducts. These adducts are suitable for the incorporation of DNA via the positive–negative charge interaction, resulting in DNA–Th–CNTs nanocomposites.

Raman spectroscopy was used to characterize those processes. Fig. 1 showed typical Raman spectra of Th (not adsorbed)

Conclusions

DNA–Th–CNTs nanocomposites were fabricated by immobilizing DNA on the surface of CNTs via Th. The nanocomposites were characterized by spectroscopic and electrochemical techniques. Th was demonstrated to be an effective mediator for DNA immobilization on CNTs. The DNA–Th–CNTs/GC electrode would be used to study the interaction of Co(bpy)33+ with DNA with a good electrochemical response and stability. The DNA–Th–CNTs/GC electrode may find use in the fields of DNA biosensors.

Acknowledgements

This work was supported by the Natural Science Foundation of China (20673057, 20773067), the Program for New Century Excellent Talents in University (NET-06-0508), and the Natural Science Foundation of Education Committee of Jiangsu Province of China (Grant No.: 06KJB150054).

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